Diamond Blackfan anemia (DBA) is a congenital anemia that develops at birth or soon after, and is due to failure of production of erythrocytes and their precursors, with normal or near normal myeloid and platelet lineages. It is inherited in about 10% of cases, mostly as an autosomal dominant. Recent genetic studies have led to the surprising identification of mutations in a ribosomal protein gene, RP219, on chromosome 19q13.2, in about 25% of both familial and sporadic cases (DBA1), and there is evidence for involvement of at least 2 other genes. Patients can remit completely on corticosteroids or may become resistant to treatment, and then require regular blood transfusions, or bone marrow transplant if a histocompatible sibling donor is available. The long term objective of this proposal is to develop preclinical data for a gene therapy protocol for severe DBA1 patients who are not eligible for matched sibling stem cell transplantation. Therefore the specific aims are (1) to identify RPS19 mutant patients by PCRT-based sequence analysis and by characterization of mutant proteins using antibodies to RPS19; (2) to further characterize the in vitro erythroid defect in these patients and then use abnormality in the erythroid progenitor cells and precursors; and (3), to "knock-in" to embryonic stem (ES) cells a mutation that has occurred independently in 6 unrelated families. The mutant ES cells will be injected into blastocysts and reimplanted into pseudopregnant females to generate chimeric animals for developing heterozygotes and breeding to homozygosity. Transmitting heterozygotes will be cross-bred to observe the consequences of mutation of both alleles in vivo. The major objective here is to create a DBA1 mouse that can be used to evaluate retrovirus and lentivirus RPS19 gene correction. Accomplishment of these goals will lead to further in vivo evaluation and a clinical protocol (not part of this project but part of the research program). In addition to the practical benefit to severely affected DBA1 patients, we hope to gain insight into how mutations in RPS19 lead to a block in the development of early erythroid cells.